The genetic deficiency of acid beta-galactosidase in humans causes two very distinct autosomal recessive diseases: GM1-gangliosidosis, a neurological disorder, and Morquio Syndrome Type B, a skeletal and connective tissue disorder. The marked phenotypic heterogeneity that exists between these two diseases is related to the differential accumulation of two natural substrates of acid beta-galactosidase, ganglioside-GM1 and keratan sulfate, in the respective diseases. The overall objective of the proposed research is to elucidate the molecular and enzymatic bases for this selective substrate accumulation. Our initial efforts will focus on defining the basic enzymology of acid beta- galactosidase and its interaction with other cellular proteins necessary for activity (i.e., the protective protein and saposin B). Natural and recombinant sources will be used for the purification of these proteins. Overexpression systems (i.e., the baculovirus based and Chinese hamster ovary systems) will be developed using our available cDNA clones for acid beta-galactosidase, protector protein and prosaposin. Detailed kinetic characterization of the pure dimeric form of acid beta-galactosidase and the acid beta-galactosidase/protector protein complex will be carried out using natural substrates in the presence and absence of the purified natural activator (saposin B) in order to determine response to the modifier, binding affinities, and turnover rates. Further definition of active site structure will be approached through detailed inhibitor studies. Coupling these in-depth kinetic studies with site-directed mutagenesis and heterologous expression experiments will lead to the identification of residues critical for the functional integrity of acid beta-galactosidase. Expressed mutations will initially include those already defined in GM1-gangliosidosis and Morquio Syndrome Type B patients. These studies will be expanded to include selected amino acid substitutions toward the goal of developing a structure/function map of acid beta-galactosidase as a rational foundation for development of diagnostic and therapeutic strategies. These experiments should provide greater insight into the molecular pathology for the widely varying phenotypes resulting from mutations in acid beta-galactosidase, as a prototype for other inborn errors of metabolism whose enzymes have several substrate specificities.